Biology subjects

Keijzer, N.

Publications and source records attributed to Keijzer, N..

3 recordsLinked to original sources

bioRxiv2024

USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that K48-linked chains are also formed on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about deubiquitination of chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA isopeptide bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis reveals that its mechanistic preference is maintained in evolution. Its kinetic mechanism results in relative enrichment of monoubiquitinated PCNA that could initially promote TLS over HR-like bypass. Taken together, these results suggest that USP1/UAF1 could be important in temporary protection of PCNA against K48- and K63-linked polyubiquitination and highlight this DUB as a potential regulator of DDT pathway choice.

biochemistry↗

bioRxiv2023

Surprising variety in the USP deubiquitinase catalytic mechanism

The USP family of deubiquitinases (DUBs) controls many ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site was so far primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine and a third residue (first critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (second critical residue) is directly adjacent to this first critical residue. Here we study the roles of these critical residues in a subset of USPs and reveal a remarkable variety in function. While USP7 relies on the first critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40 and USP48. Instead, their second critical residue is vital for catalysis. Interestingly, without their respective vital residue USP7, USP15 and USP40 can still perform nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. The unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/550302v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1f3fdbeorg.highwire.dtl.DTLVardef@197f836org.highwire.dtl.DTLVardef@28b588org.highwire.dtl.DTLVardef@a62a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisO_LIThe roles of the highly conserved critical residues in USP active sites are poorly understood. Here we show that these two residues have varying importance for catalysis between different USPs. C_LIO_LIExcept for USP7, the majority of USPs does not rely on the canonical third catalytic residue (first critical residue). Instead, the USPs tested rely primarily on the highly conserved second critical residue. C_LIO_LIIn some USPs, either critical residues can accommodate nucleophilic attack (USP7, USP40, USP15). USP1 and USP48 are unable to perform the nucleophilic attack without the second critical residue. C_LI

biochemistry↗

bioRxiv2022

Hippocampal collagen as a potential target for post-surgical treatment; effects of whole-body vibration and exercise

Peripheral surgery may evoke neuroinflammation, associated with neuronal damage and consequently mental health problems. However, anti-inflammatory treatment showed limited therapeutic efficacy. Preservation of neuron integrity during neuroinflammation, by targeting their protective collagen sheet, may provide an alternative strategy. Whole-body vibration (WBV) and exercise combine anti-inflammatory and collagen-increasing effects in the periphery. The present study aimed to explore the therapeutic efficacy of postoperative WBV and exercise on hippocampal neuroinflammation and collagen expression. Three months old male Wistar rats underwent abdominal surgery. Starting from one day after surgery, rats were submitted to WBV (10 min, once or twice daily, 30 Hz), running exercise (30 min, daily), or pseudo WBV/exercise, for two weeks. Rats were sacrificed and brain tissue was collected and processed for (immuno)histochemistry. Hippocampal microglia activity, total collagen content, and expression of fibrous and non-fibrous collagen subtypes were analysed. Surgery was associated with increased microglia activity in the CA1 area, which was only partly reversed by the interventions. Surgery specifically reduced total collagen expression in the CA1 area, which was restored by both WBV and exercise. Collagen I was absent in the hippocampal granular layers. The surgery-induced decrease in collagen III expression in the CA1 area was not affected by either WBV or exercise. However, surgery increased collagen III in the CA2 (ns), CA3 and DG. Exercise, and to a lower extent WBV, seemed to (partly) reverse this effect. Collagen IV expression was not altered by surgery, but increased by WBV. No significant effects were observed on collagen VI expression. WBV as well as exercise restored the surgery-induced declined collagen expression, while partly reversing microglia activation in the CA1 area. Moreover, effects on collagen appeared to be subtype- and region-specific, with overall similar effects of WBV and exercise. Nevertheless, the neuroprotective potential of postoperatively altered brain collagen needs further investigation.

neuroscience↗